Dear Doug,
We are doing alot of PCR sequencing. In my own experience,
sequencing symmetric PCR fragments can be difficult. The things
that have really made a difference are 1) lowering PCR primer
amount to 5 pm each (or removing PCR primers with centicon 100)
and 2) using Taq polymerase for sequencing and to cycle the
sequencing reaction using a labeled sequencing primer( in our case a
fluorescent primer). By cycling the sequencing reaction, you will
get a linear amplification of extension products which should give
plenty of sequence data. Also, with this approach you can directly
sequence symmetric PCR fragments without any purification! If you
want more info just let me know....Good-luck.
Sincerely, Sandy Koepf
Applied Biosystems Inc.
I can be contacted via ldow at apldbio.com